Bioactivity-directed isolation of antisickling compounds from

© 2020 Journal of Pharmacy & Pharmacognosy Research, 8 (6), 580-590, 2020
 ISSN 0719-4250
 http://jppres.com/jppres

 Original Article | Artículo Original

 Bioactivity-directed isolation of antisickling compounds from
 Cnidoscolus acontifolius (Mill.) I.M. Johnst leaf extract
 [Aislamiento dirigido por bioactividad de compuestos anti-sicklémicos de extracto de hoja de Cnidoscolus
 acontifolius (Mill.) I.M. Johnst]
 Mojisola C. Cyril-Olutayo*, Temitope A. Adeyemo, Ayodeji O. Oriola, Joseph M. Agbedahunsi
 Drug Research and Production Unit, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
 *E-mail: mojiolutayo@oauife.edu.ng; mojiolutayo@gmail.com

Abstract Resumen
Context: There is continuous search for therapeutic agents from Contexto: Existe una búsqueda continua de agentes terapéuticos a partir
indigenous plants that can be employed in the treatment of sickle cell de plantas autóctonas que puedan emplearse en el tratamiento de la
anemia. anemia de células falciformes.
Aims: To evaluate the antisickling potential of Cnidoscolus aconitifolius Objetivos: Evaluar el potencial anti-sicklémico del extracto de hoja de
leaf extract, determine the most active fraction, and isolate the putative Cnidoscolus aconitifolius, determinar la fracción más activa y aislar los
compounds. compuestos putativos.
Methods: Oven dried leaves of C. aconitifolius (CA) were extracted by Métodos: Se extrajeron hojas secadas al horno de C. aconitifolius (CA)
maceration in ethanol for 72 h. The extract was fractionated into n- mediante maceración en etanol durante 72 h. El extracto se fraccionó en
hexane, dichloromethane, ethyl acetate and methanol using vacuum n-hexano, diclorometano, acetato de etilo y metanol usando
liquid chromatography (VLC). The crude CA extract and fractions were cromatografía líquida de vacío (VLC). El extracto crudo de CA y las
subjected to inhibitory and reversal antisickling assays at 0.25-4.00 fracciones se sometieron a ensayos anti-sicklémicos inhibidores y de
mg/mL concentration range. Bioactivity-directed fractionation of the reversión en un intervalo de concentración de 0,25-4,00 mg/mL. El
most active fraction was done on repeated silica gel column fraccionamiento dirigido por bioactividad de la fracción más activa se
chromatography, followed by preparative thin-layer chromatography, realizó mediante cromatografía en columna de gel de sílice repetida,
and their analyses on thin-layer chromatography. The isolated seguido de cromatografía en capa fina preparativa y sus análisis en
compounds were characterized using spectroscopic methods of 1H- and cromatografía en capa fina. Los compuestos aislados se caracterizaron
13C- Nuclear Magnetic Resonance, COSY, HMBC, HSQC, and LC-MS. utilizando métodos espectroscópicos de resonancia magnética nuclear
 1H y 13C, COSY, HMBC, HSQC y LC-MS.
Results: The results showed that CA had 80.4 ± 0.15% inhibitory and 56.0
± 2.90% reversal effects at 4 mg/mL. The ethyl acetate fraction gave Resultados: Los resultados mostraron que CA tuvo 80,4 ± 0,15% de
significantly higher (p

Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

INTRODUCTION reversal antisickling activities using ultra-pure
 nitrogen gas as deoxygenating agent (Cyril-
 Medicinal plants contain organic as well as in- Olutayo and Agbedahunsi, 2015). The phytochem-
organic substances that can provide therapeutic ical screening of the ethanol leaf extracts reported
effects. They are widely used as prophylaxis for, by Chikezie et al. (2016), revealed the presence of
and treatment of, many diseases. A medicinal saponins, flavonoids, alkaloids, phlobatannins,
plant may possess a wide spectrum of effects due steroids, anthraquinones and phenols; while the
to the presence of various groups of chemical water extract showed the presence of tannins, oxa-
compounds and various microelements hence a late and cyanogenic glycosides. Numerous flavo-
preparation obtained from one plant can simulta- noid compounds most of which are kaempferol
neously be an analgesic, sedative, cardiotonic, an- and quercetin glycosides have been isolated from
ti-inflammatory, antimalarial, or anti-anemic. the leaves. However, the most active antisickling
Cnidoscolous aconitifolius (Miller) I.M. Johnson fraction and the putative compound(s) of the etha-
(family Euphorbiaceae) is known as tree spinach nol extract have not been determined, hence, this
found commonly growing in Southwestern Nige- study.
ria. It is an ornamental, evergreen plant, mostly
referred to as a deciduous plant because of its MATERIAL AND METHODS
known broad leaves. The large, 3 to 5 m tall, 32 cm
long and 30 cm wide, palmate shaped leaves are Chemicals and reagents
arranged in alternate form. C. aconitifolius (CA)
 Ethanol, n-hexane, dichloromethane, ethyl ace-
leaves are edible, commonly eaten as vegetable in
 tate and methanol (Sigma-Aldrich, St. Louis, Mis-
Nigeria (Oyagbemi et al., 2008). CA shoot and
 souri, USA) were re-distilled before use. Thin-
leaves are used as diuretic, laxative, and as stimu-
 layer chromatography (TLC) plates were pre-
lants for blood circulation and lactation. It is also
 coated Silica gel F254 (0.25 mm thickness, Darm-
used for the treatment of diabetes, acne, kidney
 stardt, Germany), while preparative TLC plates
stones and eye problem in Nigeria (Musa et al.,
 were pre-coated Silica gel G with binder (0.75 mm
2008).
 thickness, Darmstardt, Germany).
 Sickle cell anemia is a chronic hereditary ane-
mia in which the red blood cells deform in its Plant material and extraction
normal shape at low oxygen tension and become
 Fresh leaves of Cnidoscolus aconitifolius were col-
crescent shaped. It affects millions of people all-
 lected in April, 2017 at Obafemi Awolowo Junior
over the world and found to be particularly com-
 staff Quarters Road 10 with latitude 7°31'6.4488" N
mon in Sub-Saharan Africa region. Based on a re-
 and longitude 4°32'12.5484" E. The plant was iden-
search done in the year 2013, it was estimated that
 tified and authenticated by the taxonomist at the
313,000 children are born each year with SCD, 75%
 IFE Herbarium, Botany Department, OAU, Ile-Ife.
of who live in Africa (Piel et al., 2013). SCD affects
 Herbarium specimen was deposited at the IFE
about 2 to 3% of the Nigerian population of more
 Herbarium with voucher number IFE 17256. Dried
than 160 million (WHO, 2017). In South-South Ni-
 leaves (1.85 kg) were extracted by maceration in
geria, a large retrospective study in Benin-city re-
 absolute ethanol (5 L) at room temperature for 72
vealed sickle cell disease prevalence of 2.39% and
 h. Extract was evaporated to dryness in vacuo on a
a carrier rate of about 23% (Nwogoh et al., 2012).
 Buchi rotavapor and kept in the refrigerator until
Hemolysis, which is one of the symptoms of SCD,
 when needed.
results from dehydrated dense sickle cells as they
impair the microcirculation (Bartolucci et al., 2012). Collection of blood
 In our earlier studies, we demonstrated that the Fresh blood samples collected from confirmed
ethanol extract of CA possessed inhibitory and Hb SS individuals who attend routine check-ups at
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Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

the Hematology Department of the Obafemi tions were concentrated to dryness in vacuo and
Awolowo University Teaching Hospitals complex, evaluated for antisickling activities according to
Ile-Ife, Nigeria (OAUTHC) were used within 24 h Cyril-Olutayo et al. (2009).
of collection. Ethical clearance with reference
number IRB/IEC/0004553 was obtained from the Column chromatography of ethyl acetate fraction
Ethical and Research committee of the OAUTHC, The bioactive ethyl acetate fraction of CA ex-
Ile-Ife. tract (9.0 g) was adsorbed unto 25 g of silica gel
 and dry-packed on a 122 g silica gel column (60 × 5
Antisickling assay procedures
 cm) as the stationary phase. The mobile phase
 used comprised binary solvent systems of increas-
Inhibitory and reversal antisickling model
 ing level of polarity, viz: n-hexane – DCM (8:2),
 The assays were carried out using the method (6:4), (1:9); DCM – EtOAc (9:1), (6:4), (2:8) and;
of Sofowora (1979) modified by Cyril-Olutayo et EtOAc – MeOH (95:5). The eluates were bulked
al. (2009). The CA extract was tested at concentra- into seven sub-fractions (C1 - C7) based on their
tions 0.25, 0.5, 1, 2 and 4 mg/mL to determine the normal phase TLC profiles (Harborne, 1998;
optimal concentration for the bioactivity directed Hostettmann et al., 1998). They were concentrated
isolation; fractions and sub-fractions were tested at to dryness in vacuo and evaluated for antisickling
4 mg/mL concentration. Phosphate buffered saline activities (Cyril-Olutayo et al., 2009).
(0.2 mL) was used as negative control and 0.2 mL
Ciklavit® concentrate, a nutraceutical product used Column chromatography of ethyl acetate sub-fraction
in the management of SCD was employed as the C2
positive control.
 The active column bulked sub-fraction C2 (4.0
 The median effective inhibitory concentration g) was adsorbed unto 4.0 g silica gel and eluted on
was the concentration of extract which reduc- a 36 g silica gel column, using gradient solvent
es/inhibits the red blood cell sickling by 50% and systems of increasing polarity such as: n-hexane –
is calculated according to the method of Finny DCM (50:50), DCM (100%); DCM – MeOH (95:5),
(1971). Percentages of inhibition are transformed (90:10), (80:20), (50:50) and; MeOH (100%). The
into probit values. The regression lines are drawn eluates were bulked into five sub-fractions C2a –
according to equation [1]. C2e, based on TLC profile, using Hex-EtOAc (8:2).
 C2c formed solid deposits and was washed with
 = + [1] 200 mL of MeOH (100%) (Harborne, 1998;
 Hostettmann et al., 1998).
 Where: a= regression coefficient; b = constant; y
= the probit; x = log10 of the concentrations Preparative TLC (PTLC) of sub-fraction C2b
(Djekoun, 2016).
 Sub-fraction C2b (72 mg) was dissolved in 3 mL
Fractionation of CA leaf extract ethyl acetate (100%) and streaked on a 20 × 20 cm
 PTLC plate. It was air-dried for 15 minutes and
 Fractionation was carried out using Vacuum
 developed in a TLC tank, using n-hexane: EtOAc
Liquid Chromatography method (Harborne, 1998;
 (7:2) solvent system. The developed plate was
Hostettmann et al., 1998). Seventy-five grams of
 dried and visualized under the UV light (254 nm
CA extract was adsorbed unto 75 g of silica gel
 and 366 nm). Five bands were separately
and dry-packed on a 225 g silica gel Vacuum Liq-
 scrapped, filtered with EtOAc (100%), and concen-
uid Chromatography (VLC) set up as stationary
 trated to dryness in vacuo. They were analysed on
phase. Solvents (mobile phase) were introduced
 TLC using Hex-EtOAc (9:1) (Harborne, 1998;
based on increasing polarity starting from n-
 Hostettmann et al., 1998).
hexane (3.45 L), dichloromethane (3.65 L), ethyl
acetate (4.04 L) and lastly methanol (3.75 L). Frac-

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Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

Spectroscopic analysis which is equivalent to 5.1% yield.
 The isolated compounds were characterized on
 Antisickling properties of the Cnidoscolus
a 400 MHz Agilent-NMR; 300 and 600 MHz
 aconitifolius crude extract and fractions
Bruker NMR Spectrometer, where information on
1H, 13C, DEPT-135, COSY, HSQC, and HMBC were Concentration dependent inhibitory and rever-
recorded (Elufioye et al., 2016). sal activities were observed, with the highest activ-
 ities at 4 mg/mL, for CA crude extract on Hb SS
Liquid Chromatography-Mass Spectrometry red blood cells using sodium metabisulphite as
 deoxygenation agent (Table 1, Fig. 1). The IC50 of
 Reversed‑phase chromatography was per-
 the crude extract of CA was 2.17 mg/mL for the
formed on a Phenomenex Gemini‑NX 5 µm C18
 inhibitory and 3.19 mg/mL for the reversal activi-
column (100 mm × 4.6 mm) using an Alliance
 ties.
HPLC system 2695 (Waters). The column tempera-
ture was set at 25°C and the variable wavelength The ethyl acetate fraction of CA extract gave the
UV‑Vis detector was set at 220 nm. An elution highest inhibitory and reversal antisickling prop-
gradient was applied with solvent A (0.1% formic erties. These activities are significantly higher
acid in water) and solvent B (0.1% formic acid in (p

Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

 Table 1. Inhibitory and reversal effects of the crude ethanol extract of Cnidoscolus
 aconitifolius on Hb SS blood cells.
 Concentration (mg/mL) % Inhibition % Reversal
 0.25 19.22 ± 9.75 19.69 ± 0.17
 0.50 20.67 ± 3.58 21.37 ± 1.64
 1.00 23.06 ± 0.21 22.60 ± 5.29
 2.00 52.43 ± 0.61 45.00 ± 3.38
 4.00 80.40 ± 0.15* 56.00 ± 2.90*
 Ciklavit® 59.83 ± 0.30 56.57 ± 0.20
 Results were shown as mean ± standard error of mean of the analysed values (n=3). *P

Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

 Table 4. Percentage inhibition and reversal activities of sub-fractions C1 – C6.
 Fraction code % Inhibition % Reversal
 C1 40.93 ± 0.03b 25.99 ± 3.01b
 C2 69.65 ± 1.02d 82.75 ± 2.11d
 C3 52.98 ± 3 02c 29.22 ± 1.12b
 C4 49.94 ± 3.06c 52.24 ± 3.36c
 C5 40.11 ± 2.31b 53.70 ± 1.08c
 C6 24.44 ± 3.12a 17.54 ± 0.09a
 Data are expressed as mean ± SEM (n=3), analyzed using One-way ANOVA, followed by Student-
 Newman-Keul’s posthoc test. Values with different alphabets in superscripts are significant (p

Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

 A B

 Figure 2. (A) inhibition and (B) reversal of Hb SS red blood cells by C2 subfraction of CA extract (4 mg/mL).
 Magnification 400×.

 Figure 3. Inhibitory effect of pure compound T1 (4
 mg/mL) on Hb SS red bloods cells. Magnification 400×.

Structure elucidation of isolated compounds T1 13C
 NMR (100 MHz, CDCl3) δ ppm : 37.24 (C-1),
and T2
 31.66 (C-2), 71.81 (C-3), 42.32 (C-4), 140.75 (C-5),
Compound T1 121.72 (C-6), 29.17 (C-7), 39.77 (C-8), 56.06 (C-9),
 36.14 (C-10), 24.29 (C-11), 33.93 (C-12), 45.83 (C-13),
 TOF MS ES+ (m/z, % abundance): 369.6360 56.76 (C-14), 19.06 (C-15), 23.07 (C-16), 36.49 (C-17),
[M]+ (28%) consistent with the molecular formula 50.13 (C-18), 18.81 (C-19), 31.90 (C-20), 28.24 (C-21),
C26H42O (exact mass = 370.32), m/z 312.6073 [M- 26.08 (C-22), 21.08 (C-23), 19.44 (C-24), 19.34 (C-25),
58]+ (60%), m/z 274.5071 [M+] (100%), m/z 11.96 (C-26).
222.3815 [M – 148]+ (19%), m/z 182.2201 [M – 188]+
(28%), m/z 167.3177 [M – 203]+ (10%). Compound T2
 1H NMR (400 MHz, CDCl3) δ ppm: 0.68 (3H, s, Molecular formula: C21H36
H-25), 0.85 (3H, bd, H-23), 0.92 (3H, d, H-26), 1.02
 TOF MS ES+ (m/z, % abundance): 289 [M+H]+
(3H, s, H-24), 2.01, (H, t, H-4), 3.51 (H-OH, m, H-3),
 (5%) consistent with the molecular formula C21H36,
5.33 (H, brs, H-12).
 m/z 274.5071 (M+) (100%, base peak), m/z

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Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

222.3815 [M – 148]+ (19%), m/z 182.2201 [M – 188]+ antisickling effect of CA confirmed the report by
(28%), m/z 167.3177 [M – 203]+ (10%). Sarmiento-Franco et al. (2003) of the ameliorative
 1H effects of various extracts of C. aconitifolius on
 NMR (400 MHz, CDCl3) δ ppm: 0.85 (3H, t,
 anemia and osmotic fragility. The crude extract of
H-19), 0.98 (3H, s, H-20), 1.10 (3H, s, H-21)
 CA had been reported to contain phytochemicals
 13C NMR (400 MHz, CDCl3) δ ppm: 37.29 (C-1), such as alkaloids, carbohydrates, aminoacids, sap-
24.47 (C-2), 29.25 (C-3), 29.45 (C-4), 25.20 (C-5), onins, tannins, flavonoids, terpenoids, glycosides,
29.70 (C-6), 32.68 (C-7), 22.73 (C-8), 27.98 (C-9), steroids (Orji et al., 2016), amino acids such as glu-
36.50 (C-10), 22.63 (C-11), 39.37 (C-12), 42.40 (C-13), tamic acid, lysine, histidine, alanine, aspartic acids
33.66 (C-14), 25.20 (C-15), 29.93 (C-16), 31.93 (C-17), amongst others were reported to be present in the
24.87 (C-18), 14.12 (C-19), 21.40 (C-20), 19.75 (C-21). leaf extracts of CA, some of which have been im-
 plicated in antisickling activities (Markus et al.,
DISCUSSION 2016). Tannins have anti-inflammatory properties
 The antisickling assays results showed that, the and its presence in the plant could quicken the
crude ethanol extract of Cnidoscolus aconitifolius healing of wounds. Saponins have both hemolytic
was very active with 80.40 ± 0.15% inhibitory and and cholesterol binding activities (Stray, 1998)
56.0 ± 2.9% reversal activity at 4 mg/mL (Table 1 while the presence of phenol in the plant suggests
and Fig. 1) with sodium metabisulphite as deoxy- the ability to block specific enzymes that cause
genating agent. This finding is in accordance with inflammation (Okwu, 2001). Phenolic compounds
our earlier study where deoxygenation was in- are also known for their antisickling properties.
duced by nitrogen gas (Cyril-Olutayo and Agbe- Flavonoids are antioxidants and free radical scav-
dahunsi, 2015). The ethyl acetate fraction among engers and also show anti-allergic and anti-
the other fractions was the most active with 68.03 ± inflammatory, and antimicrobial properties
4.32% inhibition and 61.42 ± 6.18% reversal. The (Owolagba et al., 2009).
activity was concentration dependent as activity Bioactivity-guided fractionation of the most ac-
increased with increased concentration. The antis- tive ethyl acetate fraction yielded sub-fractions C1-
ickling properties of the ethyl acetate fraction was C6. C2 with the highest antisickling properties
significantly higher than that of Ciklavit® (positive (Table 4 and Fig. 2) led to the isolation of two
control) (Tables 2 and 3). This suggested that the compounds, T1 and T2 (see spectra in supplemen-
antisickling compounds are moderately polar. The tary data).

 Figure 4. Structure of compound T1 isolated from the ethyl acetate (C2 column) fraction of C. aconitifolius.
 Chemical Formula: C26H42O. Exact Mass: 370.32.
 6a,11,12b,14b-Tetramethyl-1,2,3,4,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicen-3-ol

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Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

 14.12

 24.87
 15.9 23.6 12.5
 19.75
 40.0
 39.37 53.1 H
 31.93 20.8
 22.63 16.1
 42.40 48.4
 21.40 38.8 30.6
 29.93 52.7 56.4
 37.29 27.98
 22.3
 24.47
 33.66 25.20 36.5 35.7 24.0
 36.50 22.73
 H H
 26.9 32.1
 29.25 32.68 47.7
 25.20

 29.45
 28.7 28.7
 29.70

 Figure 5. Structure of compound T2 isolated from the ethyl acetate fraction of C. aconitifolius.
 17-Ethyl-hexadecahydro-10,13-5β-regnane dimethyl-1H-cyclopenta[β]phenanthrene (T2) (PubChem, 2019).

 TLC profile of T1 showed it was UV-active and ster, 1998; Pavia et al., 2001). The NMR spectra (1H
turned purple with vanillin/H2SO4 spray, which and 13C) of T1 were compared with campesterol
suggested it to be an unsaturated terpenoidal reported by Choi et al. (2007).
compound. T2 also gave purple colouration but
 The mass spectrometry of T1 showed a molecu-
was UV-inactive, which suggested a saturated
 lar ion peak at m/z 369.6360 [M]+ (28%) consistent
terpenoidal compound. T1 exhibited an 83.6 ±
 with the molecular formula C26H42O. Thus, T1 was
0.11% inhibitory effect (Fig. 3). It is an antisickling
 identified as tetramethyl icosahydropicen-3-ol (see
agent which can be suitable as a drug or a lead
 spectra in supplementary data).
compound in the synthesis of antisickling drugs.
 The 1H NMR spectrum of T2 showed three
 The 1H NMR spectrum of T1 showed four
 shielded methyl groups which comprised one tri-
shielded methyl protons (CH3), which comprised
 plet at δH 0.85 ppm and two singlet signals at δH
three singlets and one doublet signals at δH 0.68,
 0.98 ppm and 1.10 ppm. 13C NMR spectrum of T2
0.85, 1.02 and 0.92 ppm respectively, thus depicts
 showed a total twenty-one signals, which repre-
an α-amyrin class of terpene (Pavia et al., 2001).
 sented twenty-one carbon atoms. The DEPT 135
The broad singlet signal at δH 5.33 ppm confirmed
 experiment showed eleven CH2, and eight CH and
the presence of a terminal olefinic proton (i.e. H –
 CH3 signals. The absence of two carbon signals at
C = C – X). While the multiplet signal at δH 3.51
 δC 36.50 ppm and 42.40 ppm on the DEPT 135
ppm confirmed the presence of a methine proton
 spectrum confirmed two quaternary carbon atoms.
(CH) directly attached to a hydroxyl (OH) group.
 The general pattern observed in the DEPT 135 ex-
 The 13C NMR spectrum of T1 showed twenty- periment suggested a pregnane skeleton (see spec-
six signals representing the number of carbon at- tra in supplementary data) (PubChem, 2019).
oms. These comprised four shielded methyl
 The mass spectrometry of T2 showed a molecu-
groups at δC 11.96, 19.34, 19.44 and 21.08 ppm. The
 lar ion peak at m/z 289 consistent with the molec-
methylene carbon atoms resonated between δC
 ular formula C21H36. A possible loss of methyl
18.81-37.24 ppm, while the signals between δC
 group at C-21 position was accounted for by the
31.90-71.81 ppm represents the methine carbons.
 largest fragment ion at m/z 274.5071 (see spectra
All of these gave an indication that the isolate is a
 in Annex), which represented the base peak (M+).
terpenoid. The deshielded carbon signal at δC 71.81
 Upon consideration of the above spectra data and
ppm was due to an attachment of hydroxyl group.
 in comparison with literature report (PubChem,
While the two further deshielded signals at δC
 2019), T2 was characterize as 5β-pregnane. This
121.72 and 140.75 ppm was due to an unsaturation
 marks the first report of the two compounds in C.
(double bond) of a cyclic ring caused by a pi elec-
 aconitifolius leaf.
tron-rich carbon (olefinic) (Silverstein and Web-
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Cyril-Olutayo et al. Antisickling compounds from Cnidoscolus acontifolius leaf extract

CONCLUSIONS Johnst. on Hbs red blood cells in vitro. Niger J Nat Prod
 Med 19: 115–121.
 The study has established the ethyl acetate frac- Djekoun M, Berrebbah H, Saib, Djebar MR (2016)
tion of C. aconitifolius leaves as the most active an- Determination of median effective inhibitory
tisickling fraction. Bioactivity-guided fractionation concentration of three fungicides widely used for
 treatment of wheat on the target pest Fusarium spp. Res J
led to the isolation of two compounds T1 and T2 Environ Toxicol 10: 109–114.
identified as tetramethylicosahydropicen-3-ol and Elufioye TO, Obuotor EM, Agbedahunsi JM (2016)
5β-pregnane. T1 exhibited an 83.6 ± 0.11% inhibi- Cholinesterase inhibitory activity and structure
tory effect against the production of sickle cells in elucidation of a new phytol derivative and a new
vitro, hence, could be lead compound in the search cinnamic acid ester from Pycnanthus angolensis. Rev Bras
 Farmacogn 26(4): 433–437.
for candidate drugs for the management of sickle
cell disease. Finny DJ (1971) Probit Analysis. London, UK: Cambridge
 University Press.
 Harborne AJ (1998) Phytochemical Methods: A Guide to
CONFLICT OF INTEREST
 Modern Techniques of Plant Analysis. 3rd ed. Springer
 The authors declare no conflict of interest. Netherlands.
 Hostettmann K, Marston A, Hostettmann M (1998)
ACKNOWLEDGMENTS Preparative chromatography techniques: Application in
 natural product isolation. Springer-Verlag Berlin
 The authors acknowledge Prof. Satyajit Sarker, School of Heidelberg.
Pharmacy and Biomolecular Sciences, Faculty of Science, Khan KM, Nahar L, Mannan A, Arfan M, Khan GA, Al-Groshi
Liverpool John Moores University, Liverpool, United King- A, Andrew Evans A, Dempster NM, Ismail FMD, Sarker
dom for the postdoctoral opportunity and spectroscopic anal- SD (2018) Liquid chromatography mass spectrometry
ysis of isolated compounds. This research work did not re- analysis and cytotoxicity of Asparagus adscendens roots
ceive any grant from funding agencies in the public, commer- against human cancer cell lines. Pharmacogn Mag
cial, or not-for-profit sectors. 13(suppl. 4): S890–S894.
 Markus V, Paul AA, Yahaya J, Zakka J, Yatai KB, Oladeji M
SUPPLEMENTARY DATA (2016) An under exploited tropical plant with promising
 economic values and the window opportunities for
 Supplementary data associated with this article can be found at
http://jppres.com/jppres/pdf/vol8/jppres20.864_8.6.580.suppl.pdf researchers: Cnidoscolus aconitifolius. Am J Food Sci Nutr
 Res 3(6): 177–187.

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 AUTHOR CONTRIBUTION:

 Contribution Cyril-Olutayo MC Adeyemo TA Oriola AO Agbedahunsi JM

 Concepts or ideas x

 Design x x

 Definition of intellectual content x x x

 Literature search x

 Experimental studies x x x

 Data acquisition x x

 Data analysis x x

 Statistical analysis x

 Manuscript preparation x

 Manuscript editing x x x

 Manuscript review x x x x

Citation Format: Cyril-Olutayo MC, Adeyemo TA, Oriola AO, Agbedahunsi JM (2020) Bioactivity-directed isolation of antisickling compounds
from Cnidoscolus acontifolius (Mill.) I.M. Johnst leaf extract. J Pharm Pharmacogn Res 8(6): 580–590.

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